Genetic code expansion has allowed the site-specific incorporation of more than a hundred unnatural amino acids into proteins. However, the utility of these approaches may be limited by the efficiency with which unnatural amino acids are incorporated into proteins. The efficient, co-translational, site-specific incorporation of unnatural amino acids into proteins will enable emerging approaches for creating site-specifically modified recombinant proteins (1, 2), as well as strategies to precisely control and image protein function in vivo (3, 4), and many other approaches in which designer unnatural amino acids are used to control or report on protein function.
Orthogonal tRNA synthetase/tRNA pairs direct the incorporation of unnatural amino acids, most commonly in response to the amber stop codon (UAG). The efficiency of unnatural amino acid incorporation is defined both by i) the intrinsic efficiency with which the orthogonal synthetase/tRNA pair enables translational elongation in response to a UAG codon in the A site of the ribosome, and ii) the efficiency with which release factors compete with the aminoacylated orthogonal tRNACUA to terminate protein synthesis. The pyrrolyl-tRNA synthetase (PylRS)/tRNACUA pair is arguably the most useful pair to be developed for genetic code expansion because i) it is orthogonal in a range of hosts including E. coli, yeast, mammalian cells, C. degans and D. melanogaster, ii) PylRS does not recognize the common 20 amino acids, iii) PylRS does not recognize the anticodon of its cognate tRNACUA, iv) the active site of PylRS accommodates a range of unnatural amino acids bearing useful functional groups without the need for directed evolution, v) the active site of PylRS can be evolved to recognize structurally diverse unnatural amino acids bearing a range of useful functional groups in E. coli and vi) the synthetase variants discovered in E. coli may be used in diverse eukaryotic hosts, where directed evolution of synthetases is challenging to implement (5).
Unnatural amino acid incorporation is currently less efficient in eukaryotic cells than in E. coli. The efficient, site-specific introduction of unnatural amino acids into proteins in eukaryotic cells is an outstanding challenge in realizing the potential of genetic code expansion approaches. Addressing this challenge will allow the synthesis of modified recombinant proteins in eukaryotic cells and augment emerging strategies that introduce new chemical functionalities into proteins to control and image their function with high spatial and temporal precision in eukaryotic cells.
The present invention seeks to address this need.